Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Full citations for those references that are numbered can be found at the end of the specification. Each citation is incorporated herein as though set forth in full.
Functional genomic studies have been hampered by the inability to uniformly express and purify biologically active proteins in heterologous expression systems. Despite the use of identical transcriptional and translational signals in a given expression vector, expressed protein levels have been observed to vary dramatically (5, 7). For this reason, several strategies have been developed to express heterologous proteins in bacteria, yeast, mammalian and insect cells as gene-fusions.
The expression of heterologous genes in bacteria is by far the simplest and most inexpensive means available for research or commercial purposes. However, some heterologous gene products fail to attain their correct three-dimensional conformation in E. coli while others become sequestered in large insoluble aggregates or “inclusion bodies” when overproduced. Major denaturant-induced solubilization methods followed by removal of the denaturant under conditions that favor refolding are often required to produce a reasonable yield of the recombinant protein. Selection of ORFs for structural genomics projects has also shown that only about 20% of the genes expressed in E. coli render proteins that were soluble or correctly folded (36, 38). These numbers are startlingly disappointing especially given that most scientists rely on E. coli for initial attempts to express gene products. Several gene fusion systems such as NUS A, maltose binding protein (MBP), glutathione S transferase (GST), and thioredoxin (TRX) have been developed (17). All of these systems have certain drawbacks, ranging from inefficient expression to inconsistent cleavage from desired structure. Comprehensive data showing that a particular fusion is best for a certain family of proteins is not available.
Ubiquitin and ubiquitin like proteins (UBLs) have been described in the literature. The SUMO system has also been characterized. SUMO (small ubiquitin related modifier) is also known as Sentrin, SMT3, PIC1, GMP1 and UBL1. SUMO and the SUMO pathway are present throughout the eukaryotic kingdom and the proteins are highly conserved from yeast to humans (12, 15, 28). SUMO homologues have also been identified in C. elegans and plants. SUMO has 18% sequence identity with ubiquitin (28, 39). Yeast has only a single SUMO gene, which has also been termed SMT3 (23, 16). The yeast Smt3 gene is essential for viability (29). In contrast to yeast, three members of SUMO have been described in vertebrates: SUMO-1 and close homologues SUMO-2 and SUMO-3. Human SUMO-1, a 101 amino-acid polypeptide, shares 50% sequence identity with human SUMO-1/SUMO-2 (29). Yeast SUMO (SMT3) shares 47% sequence identity with mammalian SUMO-1. Although overall sequence homology between ubiquitin and SUMO is only 18%, structure determination by nuclear magnetic resonance (NMR) reveals that the two proteins share a common three dimensional structure that is characterized by a tightly packed globular fold with β-sheets wrapped around one α-helix (4). Examination of the chaperoning properties of SUMO reveals that attachment of a tightly packed globular structure to N-termini of proteins can act as nucleus for folding and protect the labile protein. All SUMO genes encode precursor proteins with a short C-terminal sequence that extends from the conserved C-terminal Gly-Gly motif. The extension sequence, 2–12 amino acids in length, is different in all cases. Cells contain potent SUMO proteases that remove the C-terminal extensions. The C-terminus of SUMO is conjugated to E amino groups of lysine residues of target proteins. The similarity of the enzymes of the sumoylation pathway to ubiquitin pathway enzymes is remarkable, given the different effects of these two protein modification pathways. Sumoylation of cellular proteins has been proposed to regulate nuclear transport, signal transduction, stress response, and cell cycle progression (29). It is very likely that SUMO chaperones translocation of proteins among various cell compartments, however, the precise mechanistic details of this function of SUMO are not known.
Other fusions promote solubility of partner proteins presumably due to their large size (e.g., NUS A). Fusion of proteins with glutathione S-transferase (GST) or maltose binding protein (MBP) has been proposed to enhance expression and yield of fusion partners. However, enhanced expression is not always observed when GST is used as GST forms dimers and can retard protein solubility. Another problem with GST or other fusion systems is that the desired protein may have to be removed from the fusion. To circumvent this problem, protease sites, such as factor X, thrombin or Tev protease sites are often engineered downstream of the fusion partner. However, incomplete cleavage and inappropriate cleavage within the fusion protein is often observed. The present invention circumvents these problems.